Generation Of Colony-forming Cells Research Articles

Depleting Autologous Activated CD4+ t Lymphocytes Increased Generation of Colony-Forming-Cells(CFC) in Patients with Myelodysplastic Syndromes

Objective To investigate how autoimmune mechanism playing a role in generation of colony-forming-cells(CFC), bone mononuclear cells(BMNC) from MDS were removed of autologous activated CD4+ T cells in vitro and cultured to find out effect of T cells on MDS hemopietic progenitor.Methods BMNC from 25 patients with low-grade MDS and 5 normal donors were depleted of CD4+CCR5+ T lymphocytes using magnetic sorting. Depleted and plused CD4+CCR5+ T BMNC were seeded onto methycellulose and the correlation of colony-forming-cell (CFC) number and the polarization of T cells were analyzed, the generation of CFC, the immunophenotype and the clonal cells(which had cytogenetic markers detected by FISH), was compared respectively.Results ¢Å The capacity of BMNC from 5 healthy donors to generate CFC remained unchanged in the CD4+CCR5+ T lymphocyte-depleted and lymphocyte-plused BMNC. In contrast, cultures initiated with CD4+CCR5+ T lymphocyte-depleted BMNC from patients with low-grade MDS exhibited significantly increased generation of CFC compared with the corresponding lymphocyte-plused cultures, but the lymphocyte-plused cultures had no generation of CFC. ¢Æ The number of CFU-E from the CD4+CCR5+ T lymphocyte-depleted BMNC from patients with low-grade MDS showed significantly correlation with the percentage of Th1 (r=0.52, p≤¼ 0.05), but had no correlation with the percentage of Tc1 and the rate of Th1/Th2 and Tc1/Tc2 (p >0.05); The number of CFC, CFU-G and CFU-GE had no correlation with the polarization of T lymphocyte (P >0.05). ¢Ç The percentage of CD34 in bone nucleated cells of low-grade MDS was higher than that in healthy donors(1.8% vs. 1.0%, P >0.05), that of CD33 in nucleated cells of low-grade MDS was significantly higher than that in healthy donors[(20.3±5.8)% vs.(13.8±1.8)%(P≤¼0.05)], and that of CD13 in nucleated cells of low-grade MDS was significantly higher than that in healthy donors[(21.1±6.4)% vs. (11.6±1.8)%(p<0.05)]. After cultivation, the percentage of CD34 in low-grade MDS nucleated cells decreased to 1.4%(P >0.05), that of CD33 decreased to (12.1±3.7)%(p<0.05), and that of CD13 decreased to (17.1±5.4)%(p<0.05), but the percentage of CD34, CD33 and CD13 had no significantly changed in healthy donors between pre-culture and post-culture. ¢È FISH analysis in 6 patients revealed that +8 clone was increased(from 51% to 61%), but 20q- and -7 clone cell had no significantly changed.Conclusion In certain subtypes of MDS, selectedly removement of autologous activated CD4T cells can increase the generation of colony-forming-cells(CFC) in vitro, and improve the differentiation of MDS medullary system, but the increased CFC consisting of residual normal hemopoiesis or conal hemopoiesis were still unconcluded.

Autologous lymphocytes inhibit hemopoiesis in long-term culture in patients with myelodysplastic syndrome.

The current therapy of myelodysplastic syndrome (MDS) is unsatisfactory and comprises mainly supportive treatment or antileukemic chemotherapy. Recent studies about successful immunosuppressive therapy suggest an autoimmune mechanism in subtypes of myelodysplastic syndrome. To investigate this hypothesis, bone marrow mononuclear cells (MNC) from 15 patients with low-grade MDS, refractory anemia, and refractory anemia with ringed sideroblasts (RA and RARS), and from 7 normal donors were depleted of CD2(+), CD5(+), and CD7(+) lymphocytes using magnetic cell sorting. Depleted and nondepleted MNC were seeded onto irradiated allogeneic bone marrow stroma and the generation of colony-forming-cells (CFC), the clonal origin of hemopoietic progenitor cells in long-term bone marrow culture (LTC), was compared. The capacity of MNC from 7 healthy donors to generate hemopoiesis remained unchanged in the lymphocyte-depleted LTC. In contrast, cultures initiated with lymphocyte-depleted MNC from patients with RA and RARS exhibited a significantly increased generation of CFC compared with the corresponding nondepleted cultures. Microsatellite analysis in 6 patients revealed that a significantly increased number of CFC grown in lymphocyte-depleted LTC showed no allelic loss, suggesting an outgrowth of normal hemopoietic cells. These results provide a rationale for the recently described successful treatment of MDS with immunosuppressive therapy. We suggest that in certain subtypes of MDS the residual normal hemopoiesis is suppressed by autoimmune mechanisms, eventually allowing the expansion of the abnormal clone.

Increased apoptosis of bone marrow CD34(+) cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus.

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34(+) cells, compared with the control group, reflecting the decrease of both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells. Patient CD34(+)/Fas(+) but not CD34(-)/Fas(+) cells were significantly increased. Apoptotic (7AAD(dim)) cells were higher among CD34(+)/Fas(+) than among CD34(+)/Fas(-) cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34(+)/Fas(+) cells. These findings are suggestive of a Fas-mediated apoptosis accounting for the low CD34(+) cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E), compared with the control group, and that the generation of colony-forming cells in long-term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34(+) cells were increased, apoptotic CD34(+)/Fas(+) cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

Endothelial cell support of hematopoiesis is differentially altered by IL-1 and glucocorticoids.

We investigated the ability of endothelial cells (EC) to support hematopoiesis in contact and non-contact cocultures with isolated CD34+ or CD34/CD38low cells. In the absence of exogenous cytokines, umbilical vein EC (HUVEC) efficiently support proliferation of hematopoietic cells and generation of colony-forming cells (CFC). Cytokines (IL-6, LIF, G-CSF, GM-CSF, M-CSF, but not IL-1, IL-3, IL-7) were detected in HUVEC coculture supernatants. Neutralization of these cytokines profoundly inhibited the ability of EC supernatants to support the differentiation of hematopoietic progenitors and led to an accumulation of immature cells. Contact cocultures were significantly more efficient than non-contact cocultures. The expanded cell population essentially belonged to the myeloid and monocytic lineages. Contact cocultures generated cells expressing the CD61 or CD41 antigens. Interleukin-1alpha (IL-1alpha) augmented EC capacity to support hematopoiesis, this property resulting from the upregulation of cytokine expression. Glucocorticoids (GC) reduced this capacity by downregulating the biosynthesis of cytokines by EC and not by a direct effect on the progenitor cells. EC from the bone marrow microvasculature (BMEC) support the proliferation and the differentiation of hematopoietic progenitors. Synergistic increase in progenitor cells expansion and generation of CFC occurred when EC cocultures were added with exogenous cytokines. Supernatants of IL-1alpha-stimulated EC potentiated the effects of an association of IL-1, IL-3, IL-6, LIF, SCF, Flt3-ligand, TPO, G-CSF, GM-CSF, M-CSF and IL-11 on the proliferation of hematopoietic progenitors suggesting that EC may produce other soluble growth factors potentiating the action of the above set of cytokines.

Regulation of colony forming cell generation by flt-3 ligand.

The recently cloned ligand for the flt-3/flk-2 receptor was examined for its effect on colony formation by subpopulations of CD34+ cells including the least mature CD34+lin-CD38- small-medium lymphocyte-sized cell population. Flt-3 ligand (flt-3l) had little or no effect when added alone to cells. Isolated CD34+lin+ cells formed increased numbers of colony-forming cells (CFC) when flt-3l was added together with IL-3, IL-6, G-CSF, GM-CSF or c-kit ligand (KL), or with the combination of IL-3 and KL. Significant increases in CFC formation from CD34+lin- cells were consistently seen when flt-3l was added to the IL-3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34+lin- cells in liquid cultures showed that cultures containing IL-3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL-3, KL and flt-3l produced few CFC past 2 weeks of culture. Flt-3l alone or the combination of IL-3 and KL did not stimulate significant growth of CD34+lin-CD38- small-medium lymphocyte-sized cells, although these cells reproducibly generated CFC when grown in the combination of IL-1 beta, IL-3, IL-6, G-CSF, GM-CSF and KL. Addition of flt-3l to either IL-3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt-3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34+ cells.

Hematopoietic Precursors Resistant to Treatment With 4-Hydroperoxycyclophosphamide: Requirement for an Interaction With Marrow Stroma in Addition to Hematopoietic Growth Factors for Maximal Generation of Colony-Forming Activity

AbstractWe tested the ability of CD34+lin– precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin– cells to form CFC. However, generationof CFCfromCD34+lin– cells treated with 60 µg/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin– 4HC-treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin– precursors was also further defined as CD38–. These studies show that the generation of CFC from the 4HC-resistant, highly immature population of CD34+lin– cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.

The generation of colony-forming cells (CFC) and the expansion of hematopoiesis in cultures of human cord blood cells is dependent on the presence of stem cell factor (SCF)

We have analyzed the effect of stem cell factor (SCF), alone or in combination with other growth factors, on the generation of colony-forming cells (CFC) and on the expansion of hematopoiesis in vitro from light density, soybean agglutinin-, CD34+ cord blood cells under serum-deprived conditions. The growth factors were either added only once at the onset of the culture or added every few days when the cultures were demidepopulated and refed with fresh medium. No growth factor, alone, generated CFC or expanded hematopoiesis under these conditions. However, SCF, in combination with interleukin 3 (IL-3) or with "late-acting factors" (granulocyte colony-stimulating factor (G-CSF) or erythropoietin (Epo)), generated large numbers of mature cells as well as CFC. The number of CFC generated depended on the refeeding procedure adopted. In cultures never refed, the CFC numbers increased from < 160 CFC/culture at day 0 to > 3000 CFC at day 10. The CFC numbers stayed above the input levels for 25 days before declining. Almost no CFC were detectable after one month. In contrast, in cultures regularly refed, CFC were detectable for at least 40 days. The lineages of the mature cells and the types of CFC generated varied with the different growth factors. In the presence of SCF plus IL-3, erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (GM-CFC) were generated and erythroid as well as myelomonocytic precursors were present among the differentiated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological characteristics of recombinant rat and human stem cell factor

Recombinant stem cell factor (SCF) is the ligand for c-kit, a cell surface receptor with tyrosine kinase activity. In vitro, SCF alone stimulates little proliferation of bone marrow hematopoietic colony-forming cells (CFC). However, SCF enhances both the number and size of colonies formed in response to various cytokines. Importantly, SCF also enhances the in vitro generation of CFC from purified bone marrow precursor populations studied at a clonal level. In vivo administration of human SCF to normal baboons enhances the number of circulating granulocyte-macrophage colony-forming units (CFU-gm) and erythroid burst-forming units (BFU-e) 10- to 100-fold. CFU-mix and high proliferative potential (HPP)-CFC do not normally circulate, but after SCF treatment, seven out of seven baboons had detectable circulating levels of these progenitors. We have examined the effects of rat SCF on hematopoietic recovery after otherwise lethal total body irradiation. The LD50 for the control BDF1 mice used in these studies is between 1000–1050 cGy and 1250–1350 cGy for mice receiving only three injections of rat SCF. The most significant contribution to the radioprotective effect is due to pretreatment with rat SCF. We examined the recovery of bone marrow progenitors and mature peripheral blood cells relative to irradiated control mice. The rate of recovery of bone marrow progenitors in the SCF-treated mice was faster than in the controls, with a small difference in the total WBC between the two groups during the time (10 days post irradiation) when 90% of control mice and 0% of the SCF-treated mice were dead. There was a significant difference in platelet counts and neutrophils in the marrow between controls and SCF-treated mice which may have contributed to the death of control mice. Additional effects on other organ systems are being examined.

Long-term cultures of chicken bone marrow cells

We report an adaptation to cultures of chicken bone marrow cells of the Dexter culture technique for obtaining long-term hemopoiesis in vitro. Cells were seeded in DMEM supplemented with fetal calf serum (20%) and hydrocortisone (10 −6 M) with or without chicken serum (1%). Cultures were incubated at 37 °C and fed every 2 weeks. An adherent cell layer composed of macrophages, flbroblasts, and adipocytes became established, over which hemopoietic cells formed foci and were released into the supernatant. Granulocytes and monocytes-macrophages differentiated in a constant proportion until Week 6, whereafter differentiation became progressively restricted to the monocytic lineage. As demonstrated by the generation of colony-forming cells, hemopoiesis was maintained for either 12 or 28 weeks.